Studies on the Second Emerson Effect in the Hill Reaction in Algal Cells

This paper shows that the “second Emerson effect”¹ exists not only in photosynthesis, but also in the quinone reduction (Hill reaction), in Chlorella pyrenoidosa and Anacystis nidulans. The peaks at 650 mμ, 600 mμ, 560 mμ, 520 mμ, and 480 mμ, observed in the action spectrum of this effect in the Hill reaction in Chorella, are attributable to chlorophyll b; the occurrence of an additional peak at 670 mμ, 620 mμ, and of two (or three) peaks in the blueviolet region suggests that (at least) one form of chlorophyll a contributes to it. In analogy to suggestions made previously in the interpretation of the Emerson effect in photosynthesis, these results are taken as indicating that excitation by light preferentially absorbed by one (or two) forms of chlorophyll a (Chl a 690 + 700), needs support by simultaneous absorption of light in another form of chlorophyll a (Chl a 670)—directly or via energy transfer from chlorophyll b—in order to produce the Hill reaction with its full quantum yield. In Anacystis, the participation of phycocyanin in the Emerson effect in the Hill reaction is revealed by the occurrence, in the action spectrum of this effect, of peaks at about 560 mμ, 610 mμ, and 640 mμ; a peak at 670 mμ, due to Chl a 670, also is present.     

Accelerated publication versus usual publication in 2 leading medical journals

Background

A number of medical journals have developed policies for accelerated publication of articles judged by the authors, the editors or the peer reviewers to be of special importance. However, the validity of these judgements is unknown. We therefore compared the importance of articles published on a “fast track” with those published in the usual way.

Methods

We identified 12 “case” articles — 6 articles from the New England Journal of Medicine that were prereleased on the journal''s Web site before publication in print and 6 “fast-tracked” articles from The Lancet. We then identified 12 “control” articles matched to the case articles according to journal, disease or procedure of focus, theme area and year of publication. Forty-two general internists rated the articles, using 10-point scales, on dimensions addressing the articles'' importance, ease of applicability and impact on health outcomes.

Results

For each dimension, the mean score for the case articles was significantly higher than the mean score for the control articles: importance to clinical practice 7.6 v. 7.1 respectively (p = 0.001), importance from a public health perspective 6.5 v. 6.0 (p < 0.001), contribution to advancement of medical knowledge 6.2 v. 5.8 (p < 0.001), ease of applicability in practice 7.0 v. 6.5 (p < 0.001), potential impact on health outcomes 6.5 v. 5.9 (p < 0.001). Despite these general findings, in 5 of the 12 matched pairs of articles the control article had a higher mean score than the case article across all the dimensions.

Interpretation

The accelerated publication practices of 2 leading medical journals targeted articles that, on average, had slightly higher importance scores than similar articles published in the usual way. However, our finding of higher importance scores for control articles in 5 of the 12 matched pairs shows that current journal practices for selecting articles for expedited publication are inconsistent.A number of medical journals have developed policies for accelerated publication of articles describing findings that are judged by the authors, the editors or the peer reviewers to be particularly important and deserving of rapid dissemination. In the case of the New England Journal of Medicine, accepted articles that have “immediate clinical implications”^1,2 are occasionally prereleased on the journal''s Web site (www.nejm.org) before their official publication date, and an early press release is issued to the media. A recent high-profile example of a prereleased article is that of the RALES study that assessed the efficacy of spironolactone for congestive heart failure.^2,3The Lancet,⁴,⁵,⁶ the British Medical Journal ⁷ and CMAJ ⁸ are other medical journals that have adopted mechanisms for occasionally accelerating the peer review and printing process for articles judged to present especially important research findings needing urgent dissemination. In these journals the expedited process is referred to as “fast-track” publication. A number of other high-profile journals, including the Journal of the American Medical Association,⁹ Science¹⁰ and Nature,¹¹ have also adopted mechanisms for expedited publication, with Nature recently prereleasing on its Web site 2 articles on the molecular biology of anthrax infections.^12,13Despite the existence, and increasing profile, of these journal publication policies, no study has formally assessed the importance, methodological quality and general visibility of articles published in an accelerated manner relative to articles published in the usual manner. In this article we address these questions by asking a group of physicians to rate the importance, quality and visibility of a selection of prereleased articles from the New England Journal of Medicine and fast-tracked articles from The Lancet.     

ATG7 contributes to plant basal immunity towards fungal infection

Autophagy has an important function in cellular homeostasis. In recent years autophagy has been implicated in plant basal immunity and assigned negative (“anti-death”) and positive (“pro-death”) regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. We recently showed that Arabidopsis mutants lacking the autophagy-associated (ATG) genes ATG5, ATG10 and ATG18a are compromised in their resistance towards infection with necrotrophic fungal pathogens but display an enhanced resistance towards biotrophic bacterial invaders. Thus, the function of autophagy as either being pro-death or anti-death depends critically on the lifestyle and infection strategy of invading microbes. Here we show that ATG7 contributes to resistance to fungal pathogens. Genetic inactivation of ATG7 results in elevated susceptibility towards the necrotrophic fungal pathogen, Alternaria brassicicola, with atg7 mutants developing spreading necrosis accompanied by production of reactive oxygen intermediates. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in the atg7 mutant. We conclude that ATG7-dependent autophagy constitutes an “anti-death” (“pro-survival”) plant mechanism to control the containment of cell death and immunity to necrophic fungal infection.Key words: autophagy, ATG7, basal immunity, fungal resistance, arabidopsisPlants have evolved a bipartite plant immune system to cope with microbial infections. The first layer of defense relies on the recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PAMP-triggered immunity, PTI).^1,2 To overcome this defense strategy, successful pathogens deliver so-called effector proteins into plant cells to modify host cellular processes and to suppress immune responses to enhance virulence. The presence or activities of these microbial effectors is sensed by plant resistance proteins and triggers the second layer of defense, the effector-triggered immunity (ETI).^1,2 In contrast to PTI, ETI is most often accompanied by programmed host cell death (PCD) at the site of attempted microbial invasion; however the molecular basis of this apoptosis-like hypersensitive response (HR) is largely unknown.In recent years evidence accumulated that a non-apoptotic form of cell death called autophagy is not only involved in animal PCD and innate immunity³ but is also an important component in the plant basal immune response.⁴ Generally, autophagy (auto, meaning “self” and phagy, “to eat”) is a cytoplasmic bulk degradation process in which cellular components are targeted to lysosomal or vacuolar degradation. This process is ubiquitous in eukaryotic organisms and is considered to aid cellular survival, differentiation, development and homeostasis by nutrient recycling or removal of damaged or toxic materials.^5–7     

The Role of Binding Energy in Catalysis: the Work of William P. Jencks

Two Functional Domains of Coenzyme A Activate Catalysis by Coenzyme A Transferase. Pantetheine and Adenosine 3′-Phosphate 5′-Diphosphate (Fierke, C. A., and Jencks, W. P. (1986) J. Biol. Chem. 261, 7603–7606)William Platt Jencks (1927–2007) was born in Bar Harbor, Maine. He became interested in chemistry when he received a chemistry set for Christmas in 1934. He immediately carried out one of the experiments described in the instructions, the addition of dilute acid to a sulfide salt to produce H₂S. The experiment was so successful that his house had to be evacuated due to the smell of rotten eggs. According to Jencks, “My family and I did not find it necessary to replicate this experiment” (1).Open in a separate windowWilliam P. JencksJencks enrolled at Harvard College, intending to study chemistry. However, after taking a first year course in chemistry that “described a large number of chemical reactions, one after the other, with no indication of what was interesting about any of them” (1), he switched his major to English. Despite this change in the direction of his studies, Jencks ended up entering Harvard Medical School after his junior year because he wasn''t sure what else to do.After completing his first year of medical school, Jencks spent a summer at the Marine Biological Laboratory in Woods Hole, taking courses and doing research on lobster shell pigments with Journal of Biological Chemistry (JBC) Classic author George Wald (2). He received his M.D. in 1951 and then interned at Peter Bent Brigham Hospital in Boston. However, after a while, Jencks found medicine to be “a very broad field in which it would be difficult to obtain definitive answers to fundamental problems” (1). Wald suggested Jencks try doing research at Massachusetts General Hospital with Nobel laureate Fritz Lipmann (who was featured in a previous JBC Classic (3)). Jencks ended up spending 2 years with Lipmann, studying coenzyme A transferase, which led to his longtime interest in the physical organic chemistry of acyl transfer reactions. After leaving Massachusetts General Hospital, Jencks spent a year doing postdoctoral studies at Harvard University with Nobel laureate Robert Woodward before joining the faculty at Brandeis University in 1957, serving as assistant, associate, and then full professor of biochemistry. He retired in 1996 as professor emeritus of biochemistry.During his 39 years at Brandeis University, Jencks studied the mechanisms by which enzymes facilitate chemical reactions of molecules that are not otherwise inclined to react at a useful rate.The JBC Classic reprinted here looks at the noncovalent interactions between succinyl-CoA 3-ketoacid coenzyme A transferase and coenzyme A. In the paper, Jencks and Carol A. Fierke used a small coenzyme A analog, methylmercaptopropionate, to show that noncovalent interactions between the enzyme and the side chain of CoA are responsible for the reaction rate increase brought about by the enzyme. They report that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of substrate destabilization and rate acceleration, whereas the interaction with the 3′-phospho-ADP1 moiety provides binding energy that overcomes this destabilization and permits significant binding of acyl-CoA substrates to the enzyme. This paper helped to illuminate a striking example of the role of binding energy in catalysis.Jencks received many honors and awards for his contributions to science, including memberships in the National Academy of Sciences (1971) and the American Philosophical Society (1995) and foreign membership in the Royal Society. He also received the 1962 American Chemical Society (ACS) Award in Biological Chemistry, the 1993 American Society of Biological Chemists Award, the 1995 ACS James Flack Norris Award in Physical Organic Chemistry, and the 1996 ACS Repligen Award for Chemistry of Biological Processes.¹     

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1

Correction to: EMBO Reports (2019) 20: e47074. DOI 10.15252/embr.201847074 | Published online 6 May 2019The authors noticed that the control and disease labels had been inverted in their data analysis resulting in publication of incorrect data in Figure 1C. The corrected figure is displayed below. This change affects the conclusions as detailed below. The authors apologize for this error and any confusion it may have caused.In the legend of 1C, change from, “Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows increased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.Open in a separate windowFigure 1CCorrected Open in a separate windowFigure 1COriginal To, "Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows decreased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.In abstract, change from, “Here, we report increased IMP1 expression in patients with Crohn''s disease and ulcerative colitis”.To, “Here, we report increased IMP1 expression in adult patients with Crohn''s disease and ulcerative colitis”.In results, change from, “Consistent with these findings, analysis of published the Pediatric RISK Stratification Study (RISK) cohort of RNA‐sequencing data 38 from pediatric patients with Crohn''s disease (CD) patients revealed that IMP1 is upregulated significantly compared to control patients and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.To, “Contrary to our findings in colon tissue from adults, analysis of published RNA‐sequencing data from the Pediatric RISK Stratification Study (RISK) cohort of ileal tissue from children with Crohn’s disease (CD) 38 revealed that IMP1 is downregulated significantly compared to control patients in the RISK cohort and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.In discussion, change from, “Indeed, we report that IMP1 is upregulated in patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.To “Indeed, we report that IMP1 is upregulated in adult patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.     

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its Nα Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N^α acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in H. volcanii cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N^α acetylation. Both the “gate” deletion and Q2A substitution completely altered the N^α-acetylation pattern of α1, with the deletion rendering α1 unavailable for N^α acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N^α acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N^α-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N^α acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.Proteolysis is important in regulation and protein quality control. Energy-dependent proteases are crucial to early stages of these proteolytic events and include proteasomes, multicatalytic proteases present in all eukaryotes and archaea and in some bacteria. The catalytic component of proteasomes, the 20S core particle (CP), consists of four heptameric rings of α- and β-type subunits stacked as a barrel in an α7β7β7α7 configuration and is essential for growth of archaeal and eukaryotic cells (39, 54). The active sites responsible for peptide bond hydrolysis are formed by N-terminal Thr residues of β-type subunits and are sequestered within the central chamber of the barrel-like structure. Energy-dependent triple-A ATPases, including regulatory particle triple-A ATPases (Rpt) in eukaryotes and proteasome-activating nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins through the α-rings for degradation within the CP (39, 40).One major difference between eukaryotic and prokaryotic proteasomal CPs is in the crystal structure of the channel opening formed by the α-rings. Due to partial disorder of the α-subunit N termini, the site of substrate entry appears open at the ends of the cylinders of archaeal and bacterial CPs (e.g., CPs of Thermoplasma acidophilum, Archaeoglobus fulgidus, and Mycobacterium tuberculosis) (13, 15, 27). In contrast, X-ray structures of the CPs of yeast (14) and bovine (45) do not contain this opening. Instead the extreme N termini of the α2, α3, and α4 subunits and the loop structure of α5 fill the central pore in a gate-like structure.Evidence suggests that all CPs are gated, and the major differences observed in the state of the α-ring gate in crystal structures are not physiological. For example, the N-terminal 11 amino acids of the A. fulgidus α subunit, which are not defined by electron density in the CP structure, are more ordered in the 16S “half” proteasome precursor (13). Furthermore, cryoelectron microscopy of the M. tuberculosis CP reveals closed ends that are dependent on the first eight residues of the α-subunit and which diminish peptidolytic activity. Consistent with this, deletion of the N-terminal α-helix (Δ2-12) of the T. acidophilum CP α-subunit abolishes the need for an ATPase (i.e., PAN) in the proteasome-mediated degradation of acid-denatured green fluorescent protein-SsrA or casein (4). In addition, the conserved YDR motif thought to be important in the sterics of α-ring gating is present in all archaeal α-type subunits to date (13). Thus, prokaryotes are thought to gate the α-ring aperture of their proteasomes; however, the physiological consequences of unregulated opening of this gate have not been examined.A gated CP channel formed by the N termini of α-rings may be a general mechanism for regulating the activity of proteasomes. The rate-limiting step in proteasome-mediated protein degradation is translocation of substrates through the α-rings to the active sites contained within the β-rings of the CP (24). Gating is supported by the finding that eukaryotic CPs have no peptidolytic activity in the absence of Rpt proteins or mild chaotropic agents such as sodium dodecyl sulfate (SDS) or heat treatment (9). Furthermore, peptidase activity of the yeast CP is blocked by the N-terminal regions of the α3 subunit. Deletion (Δ2-9) or single substitution (D9A) of N-terminal residues of α3 derepresses this peptidase activity (12).An additional gating mechanism could be employed by posttranslational modifications of the N termini of the α-type subunits. The α-type subunits of CPs are modified by N^α acetylation in several eukaryotes and haloarchaea, including Haloferax volcanii (10, 16, 20, 21, 44). In yeast, N-acetyltransferase 1 (NAT1), the catalytic component of NatA, is responsible for the N^α acetylation of five of the α-type subunits (α1, α2, α3, α4, and α7). Proteasomes purified from a nat1 mutant have twofold-higher chymotrypsin-like peptidase activity in the absence of SDS compared to the wild type, suggesting that N^α acetylation enhances closure of the α-gate (21). In H. volcanii, both α1 and α2 are N^α acetylated on their initiator methionine residue with a subset of α1 not acetylated and instead cleaved by an apparent methionine aminopeptidase (16). A large-scale proteomic survey reveals N^α acetylation is common to other proteasomal α-type proteins of the haloarchaea (10). In this previous survey, the ratios of N^α-acetylated and cleaved forms of the α-type proteins were quantified by spectral counting and estimated to be around 3:1 and 4:3 for Halobacterium salinarum and Natronomonas pharaonis, respectively (10). So far, this existence of these two unique forms of α subunit N termini in the cell simultaneously (initiator methionine N^α acetylated and methionine aminopeptidase [MAP] cleaved) has only been observed in the haloarchaea.In the present study, quantitative tandem mass spectrometry (MS/MS) was used to precisely determine the ratio of the N^α-acetylated to MAP-cleaved forms of the proteasomal α1 protein in H. volcanii. In addition, site-directed mutagenesis was used to examine how the N-terminal penultimate (second) residue and N-terminal α-helix of α1 influence its N^α-acetylated state, CP activity, and cell physiology. Alterations that either fully abolished N^α acetylation or enhanced MAP cleavage of α1 (i) resulted in an increase in CP peptidase activity and (ii) rendered the cells more sensitive to hypoosmotic stress than wild type. In contrast, site-directed changes that generated a mixed population of α1 proteins in various N^α-acetylated states, yet similar N^α-acetylation profiles in CPs to wild type, had profound consequences, including (i) a substantial increase in the levels of α1 protein as heptameric rings, (ii) higher growth rate and cell yield, and (iii) enhanced tolerance of cells to thermal and hypoosmotic stress.     

Ligand Binding Reveals a Role for Heme in Translationally-Controlled Tumor Protein Dimerization

The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His⁷⁶His⁷⁷) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP''s cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca²⁺ ligand to TCTP does not alter its monomeric state; although, Ca²⁺ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP''s proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP''s role as a component of signal transduction mechanisms.     

Effects of Phenylmercuric Acetate on Stomatal Movement and Transpiration of Excised Retula papyrifera Marsh. Leaves

Effects of 10⁻³m, 10⁻⁴m, and 10⁻⁵m phenylmercuric acetate (PMA) on stomatal movement and transpiration of excised Betula papyrifera leaves were investigated. Duco cement leaf prints and transpiration decline curves were used for the analysis of stomatal condition. PMA induced stomatal closure and decreased transpiration. Stomata of leaves treated with any of the 3 PMA concentrations closed earlier and at a higher relative water content than did stomata of untreated leaves. As determined from transpiration decline curves, PMA at 10⁻³m caused an increase in apparent “cuticular” transpiration. However, the increase appeared to result largely from some PMA-poisoned stomata which remained open for prolonged periods. Considerable PMA toxicity was observed, with 10⁻³m and 10⁻⁴m concentrations causing browning of leaves. PMA treatment caused a decrease in chlorophyll content, even at a low PMA concentration (10⁻⁵m) which influenced stomatal response only slightly and did not cause evident browning of leaves. The time and degree of stomatal opening varied with stomatal size. Large stomata tended to open earlier and close later than small stomata. Hence, in Betula papyrifera stomata of various size classes were considered as physiologically different populations.     

Research letter: How should abridged scientific articles be presented in journals? A survey of readers and authors

SEVERAL SCIENTIFIC AND GENERAL MEDICAL JOURNALS publish full-length articles on their Web sites and abridged versions in their print journals. We surveyed a stratified random sample of BMJ readers and authors to elicit their preferred format for the abridged print version. Each participant received a research paper abridged in 3 different formats: conventional abridged version, journalistic version and enhanced-abstract version. Overall, 45% (95% confidence interval [CI] 42%–48%) of the respondents said they liked the conventional version most, 31% (95% CI 28%–34%) preferred the journalistic version and 25% (95% CI 22%–27%) preferred the enhanced-abstract version. Twenty-eight percent (95% CI 25%–32%) indicated that use of the journalistic format for abridged articles would very likely stop them from submitting papers to BMJ, and 13% (95% CI 11%–16%) said the use of the enhanced-abstract version would stop them from submitting to BMJ. Publishers of general medical journals who publish shortened articles should consider that authors and readers prefer a more conventional style of abridged papers.To allow critical appraisal of a scientific paper, a certain amount of space is needed to describe the methods and results. Editorial space is scarce, however, and editors struggle to meet the expectations of both authors and readers. Moreover, it seems that many readers read only parts of scientific papers. Now that electronic publishing is available, several scientific and general medical journals have adopted strategies to publish detailed articles on their Web sites, often in advance of the print journal publication, with a more concise presentation appearing in the print journal.Since 2000 most papers in BMJ have been published in their full-length form on the journal''s Web site and in an abridged form in the print journal.¹ Currently the abridgement is simply a condensed version of the full-length paper (about 30%–50% shorter). The conventional structure of scientific papers is maintained, and the original wording is hardly altered.There are many alternative forms of presenting abridged scientific information. “Serious” magazines such as The Economist use a journalistic approach, presenting the salient information at the beginning and more technical details later in the text. Medical journals such as Evidence-Based Medicine use enhanced abstracts, in which details of the results are presented (including a table or figure), and the main text — if there is any — provides context and interpretation. We performed a survey to find which format of short version — the conventional approach, a journalistic version or a version with an enhanced abstract — of scientific articles is preferred by readers and authors. We randomly sampled 2 papers each of 6 study designs (randomized controlled trial, systematic review, meta-analysis, cohort study, case–control study and qualitative study) published in conventional abridged form^2,3 in BMJ between January 2000 and June 2002. The conventional format is a condensed version of the full-length paper and contains 1300–1500 words; the structure of the paper (Introduction, Methods, Results, Discussion) is retained.³ For each paper we prepared 2 additional abridged formats: a journalistic version⁴ and an enhanced-abstract version (see the online appendix at www.cmaj.ca/cgi/content/full/172/2/203/DC1 for a sample of 3 abridgements of 1 paper). The journalistic version was based on a style typically used by “serious” newspapers, with the following subheadings: “Why we carried out this study,” “The background,” “What were the main findings?,” “How did we perform the study?” and “Why are these results important?” This format had no abstract, the paper starting instead with the main findings and conclusions in the first paragraph, followed by less salient details. The enhanced-abstract version provided detailed results in the abstract (including a table or figure), and the main body of the text was explanatory, focusing on the context and discussion.We drew a stratified random sample from 1728 UK and non-UK readers and 360 corresponding authors of a research paper published in BMJ between January 2000 and June 2002. Each participant received all 3 abridged versions of 1 of 12 research papers. The study design of the paper and the order of versions were allocated randomly to each participant.We asked the respondents to rank the 3 versions in order of preference and to indicate how strongly they felt about their preference. We also asked participants whether use of either of the journalistic or enhanced-abstract formats instead of the conventional format would prevent them from submitting papers to BMJ in the future. We offered a book voucher as an incentive and sent 2 reminders to nonrespondents.⁴Of the sample of 2088 respondents, 55 were excluded (43 had incorrect mailing addresses, and 12, being both readers and authors, were selected more than once because of overlap in the databases). Of the 2033 eligible participants, 1002 (49%) responded. The response rate was higher for authors (220/321 [68%]) than readers (782/1712 [46%]). Item nonresponse was minimal.A total of 45% (95% confidence interval [CI] 42%–48%) of the respondents (446/997) (42% of readers and 56% of authors) said that they liked the conventional abridged version most (Fig. 1). The next most preferred format was the journalistic version (31% [95% CI 28%–34%] [306/997]), followed by the enhanced-abstract version (25% [95% CI 22%–27%] [245/997]). When we stratified for location (UK v. non-UK) and audience (reader v. author), the conventional abridged version remained the preferred version, except among UK readers, who gave the 3 versions similar rankings (Fig. 1).Open in a separate windowFig. 1: Type of short version of scientific article most preferred by BMJ authors and readers, stratified for audience (authors v. readers) and geographic location (UK v. non-UK). Error bars represent the 95% confidence interval.Most respondents (87% [95% CI 85%–89%] [863/994]) liked their first choice “a lot.” Thirteen percent (95% CI 11%–15%) (127/994) liked their first choice only “a little,” and none said they “don''t like it at all.” Four respondents (0.4%) did not have an opinion.A total of 268 respondents said they were unlikely to submit a paper to BMJ in the future. Of the remaining 734 respondents, 28% (95% CI 25%–32%) indicated that use of the journalistic format for abridgement would very likely stop them from submitting papers to BMJ, and 13% (95% CI 11%–16%) said that use of the enhanced-abstract format as abridgement would.Our findings that a conventional abridged version was preferred by BMJ authors and readers and that many respondents indicated that use of journalistic and enhanced-abstract formats would prevent them from submitting future papers to the journal are important signals for editors. They indicate that contributors'' opinions should be considered when planning major changes for the presentational style of scientific papers.The main limitation of our study is the low response rate for readers (46%), despite strategies to optimize response rates.⁴ However, we believe that the information we received from readers is still useful, since we have no reason to believe that any particular selection mechanism is active other than lack of interest in the presentation of research papers.Publishers of scientific and general medical journals who use or are considering using abridgements of scientific articles should bear in mind that a conventional format of abridged papers may be the choice most likely to satisfy both readers and authors.     

Dynamic Model of Heat Inactivation Kinetics for Bacterial Adaptation

The Weibullian-log logistic (WeLL) inactivation model was modified to account for heat adaptation by introducing a logistic adaptation factor, which rendered its “rate parameter” a function of both temperature and heating rate. The resulting model is consistent with the observation that adaptation is primarily noticeable in slow heat processes in which the cells are exposed to sublethal temperatures for a sufficiently long time. Dynamic survival patterns generated with the proposed model were in general agreement with those of Escherichia coli and Listeria monocytogenes as reported in the literature. Although the modified model''s rate equation has a cumbersome appearance, especially for thermal processes having a variable heating rate, it can be solved numerically with commercial mathematical software. The dynamic model has five survival/adaptation parameters whose determination will require a large experimental database. However, with assumed or estimated parameter values, the model can simulate survival patterns of adapting pathogens in cooked foods that can be used in risk assessment and the establishment of safe preparation conditions.Combined with heat transfer data or models, microbial survival kinetics, especially of bacteria or spores, is extensively used to determine the safety of industrial heat preservation processes like canning, extant or planned. The same is true for milder heat processes such as milk and fruit pasteurization. However, survival models are also a valuable tool to assess the safety of prepared foods, especially those made of raw meats, poultry, and eggs, where surviving pathogens can be a public health issue.The heat resistance of a bacterium, or any other microorganism, is almost always determined from a set of its isothermal survival curves, recorded at several lethal temperatures. The kinetic models, which define the heat resistance parameters, may vary, but the calculation procedure itself is usually the same. First, the experimental isothermal survival data are fitted with what is known as the “primary model.” Once fitted, the temperature dependence of this primary model''s coefficients is described by what is known as the “secondary model.” When combined with a temperature profile expression, T(t), and incorporated into the inactivation rate equation, the result is a “tertiary model,” which enables its user to predict the organism''s survival curve under any static or dynamic (i.e., nonisothermal) conditions.The traditional log-linear (“first-order kinetic”) model is the best-known primary survival model, and it is still widely used in sterility calculations in the food, pharmaceutical, and other industries. Traditionally, it has been assumed that the D value calculated with this model has a log-linear temperature dependence or, alternatively, that the temperature effect on the exponential rate constant, k, the D value''s reciprocal, follows the Arrhenius equation. However, accumulating experimental evidence in recent years indicates that bacterial heat inactivation only rarely follows the first-order kinetics and that there is no reason that it should (3, 18, 29). Nonlinear survival curves can be described by a variety of mathematical models (6). Perhaps the most frequently used in recent years is the Weibullian model, of which the traditional log-linear model is a special case—see below.Regardless of the log-linearity issue, none of the above-mentioned models accounts for adaptation, the ability of certain bacterial cells to adjust their metabolism in response to stress in order to increase their survivability (2, 10, 26, 27, 28). A notable example is Escherichia coli. Its cells can produce “heat shock proteins,” which help them to survive mild heat treatments (1, 11). Other organisms, Salmonella enterica and Bacillus cereus among them, can also develop defensive mechanisms that help them to survive in an acidic environment (8, 9, 13). Whether adaptation allows the cells to avoid injury or to repair damage once it has occurred, or both, should not concern us here. (Injury and recovery, although related, are a separate issue, one which is amply discussed in the literature. Their quantitative aspects and mathematical modeling are discussed elsewhere [5].)The cells'' ability to augment their resistance is not unlimited, and it takes time for the cells to activate the protective system and synthesize its chemical elements (10, 12). Consequently, the effect of heat adaptation on an organism''s survival pattern becomes measurable only at or at slightly above what''s known as the “sublethal” temperature range. Under dynamic conditions, therefore, adaptation can be detected only when the heating rate is sufficiently low to allow the cells to respond metabolically to the heat stress prior to their destruction.Several investigators have reported and discussed the quantitative aspects of adaptation (25, 27, 28). When it occurs, adaptation is noticed as a gap between survival curves determined at low heating rates and those predicted by kinetic models whose parameters had been determined at high lethal temperatures (7, 8, 9, 27, 28). The question is how to modify the inactivation kinetic model so that it can properly account for adaptation at low heating rates while maintaining its predictive ability at high rates and clearly lethal temperatures. Stasiewicz et al. (25) have recently given a partial answer to this question. They started with the Weibullian inactivation model (see below) and assumed that its rate parameter''s temperature dependence follows a modified version of the Arrhenius equation. Using this model and experimental data for Salmonella bacteria, they showed that a “pathway-dependent model” is more reliable than a “state-dependent model.”The objectives of our work were to develop a variant of the Weibullian-log logistic (WeLL) inactivation model to account for dynamic adaptation and to demonstrate its applicability with reported adaptive survival patterns exhibited by Escherichia coli and Listeria monocytogenes, two organisms of food safety concern.     

Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge,Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.Enhanced biological phosphorus removal (EBPR) has been applied in many wastewater treatment plants to reduce the phosphorus that causes eutrophication in surface waters. EBPR employs polyphosphate-accumulating organisms (PAOs), which are enriched through alternating aerobic-anaerobic cycles (34). Since PAOs are essential for an understanding of EBPR, many candidates have been proposed as potential PAOs, such as Acinetobacter spp. (11), Tetrasphaera spp. (31), Microlunatus phosphovorus (36), Lampropedia spp. (40), and Gram-positive Actinobacteria (24). However, those organisms do not exhibit all of the characteristics of the EBPR biochemistry model. Recently developed culture-independent approaches such as PCR-clone libraries, fluorescence in situ hybridization (FISH), and microautoradiography (MAR) have highlighted an uncultured Rhodocyclus-related bacterium, “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), as one of the most important PAO candidates (2, 5, 16, 22, 23, 27, 28, 47).Numerous studies have sought to investigate uncultured “Ca. Accumulibacter” and have shown the presence of genetically and physiologically different members with a global geographic distribution (3, 9, 22, 27, 39). For example, Kong et al. (22) identified two morphologically different “Ca. Accumulibacter” cells of small cocci and large coccobacilli labeled with PAOmix (PAO462, PAO651, and PAO846) in laboratory-scale EBPR reactors. Additional results showing phenotypic and morphological diversities of “Ca. Accumulibacter” cells also existed with respect to the different roles of denitrifying PAO (DPAO) in the EBPR process (3, 9, 23). Carvalho et al. (3) detected two different morphotypes of “Ca. Accumulibacter” with different nitrate reduction capabilities. The presence of other “Ca. Accumulibacter” strains with 15% genome sequence divergence from the dominant strains in metagenomic analyses is likely to explain these morphological and phenotypic differences (12). McMahon et al. (33) suggested the use of the polyphosphate kinase (ppk) gene, which is involved in the production of polyphosphate, for a finer elucidation of “Ca. Accumulibacter” diversity. He et al. (15) grouped “Ca. Accumulibacter” strains into five distinct clades, designated clades I, IIA, IIB, IIC, and IID, using ppk gene sequence information. Flowers and colleagues (9) previously reported that “Ca. Accumulibacter” cells of clade IA had nitrate reduction activity with phosphorus uptake but that “Ca. Accumulibacter” cells of clade IIA did not.FISH-fluorescence activated cell sorting (FACS) techniques have been used for the separation of specific microbial cells from complex microbial consortia and their metabolic gene analysis (14, 46). For example, Miyauchi et al. (35) sorted PAOmix probe-labeled “Ca. Accumulibacter” cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity. In the current study, we found that four different “Ca. Accumulibacter” clades (Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4) were present in the EBPR sludge of laboratory-scale reactors supplied with acetate as the sole carbon source. We analyzed their morphological characteristics and ppk gene sequence information using a suite of FISH and FACS approaches and linked fine-scale phylogenetic diversities of “Ca. Accumulibacter” strains with their morphological characteristics and metabolic genes. This study will be useful for further genetic and physiological studies of different “Ca. Accumulibacter” clades.     

Detecting “different”: Pyrin senses modified GTPases

Pathogenic bacteria secrete effector proteins that target host cell Rho GTPases to manipulate the actin cytoskeleton. A recent study in Nature identifies the Pyrin inflammasome as a sensor of this pathogenic process.The ability to detect the presence of bacteria is crucial for life in multicellular organisms. While trillions of bacteria constitutively colonize many locations both on and inside the host, the vast majority of these pose no immediate threat. To avoid unnecessary use of resources and collateral tissue damage, the host has to carefully assess which bacteria defensive responses should be initiated. How the immune system distinguishes between pathogenic and non-pathogenic bacteria has been one of the most intriguing questions in immunology.Mammalian species use two major classes of receptors to sense microbes. Toll-like Receptors (TLRs) detect highly conserved microbial structures, such as lipopolysaccharide (LPS), lipopeptides and DNA¹. These microbial patterns are present in virtually all bacteria and, by themselves, are not informative for the immunological decision “pathogen or non-pathogen”. However, as TLRs are optimized to sense microbes in the extracellular environment (or through endocytosed “sampling” of the environment), activation of TLRs in locations that are normally sterile can inform the immune system of unwanted bacterial presence. Thus, by incorporating “location” as an additional parameter, TLR activation can be utilized to detect signs of pathogens.In contrast to TLRs, the second major class of innate sensors, which includes the Nod-like Receptors (NLRs), AIM2-like Receptors (ALRs), and Rig-I-like Receptors (RLRs), appear to have evolved to respond more selectively to pathogenic microbes². Localized in the cytosol, these receptors survey the inside of the cell for pathogen-associated molecules or activities. Upon detection of such signals, many of these receptors initiate the formation of a large multiprotein complex referred to as the inflammasome, which enables the activation of Caspase-1, pyroptosis, and the secretion of IL-1β and IL-18.Unlike harmless commensals, pathogenic bacteria employ a wide variety of mechanisms to affect the host, including attachment to or invasion of host cells and secretion of toxins. Inflammasome-inducing receptors evolved two distinct approaches to sense these various bacterial virulence strategies. The first approach involves detections of the presence of intracellular microbial patterns (“pattern-triggered immunity”); while these static signals, like TLR ligands, are often present in both commensals and pathogens, their presence in the cytosol indicates an immediate danger. Examples of “pattern-triggered immunity” in the cytosol include: NOD1- and NOD2-mediated detection of intracellular peptidoglycan; AIM2-mediated detection of DNA from replicating or lysed intracellular bacteria; NLRC4-mediated detection of intracellular flagellin and components of Type III Secretion Systems released from invading or invaded bacteria; and NLRP3-mediated sensing of cytosolic LPS via Caspase-11 (Figure 1). The second approach revolves around the detection of dynamic signals (“effector-triggered immunity”). Here, instead of sensing the pathogen directly, the immune system senses the effects of common virulence mechanisms employed by pathogens. For instance, NLRP3 detects membrane disruption by pore-forming toxins, such as aerotoxin and nigericin; NLRP1b senses proteolytic activity of the Bacillus anthracis anthrax toxin that inactivates MAP kinases; and NOD1 senses the activation of Rho GTPases by Salmonella Typhimurium^2,3.Open in a separate windowFigure 1Effector-triggered and pattern-triggered immunity in mammalian cells. Bacterial effectors (shown in red) gain access to the cytosol, where they manipulate cellular processes through pore formation in the cellular membrane (sensed by NLRP3), GTPase inactivation (sensed by Pyrin), GTPase activation (sensed by NOD1), or MAPK inactivation (sensed by NLRP1b). Bacterial patterns are released or secreted from attaching or invading bacteria, where they are detected by NLRC4 (sensing flagellin and components of the Type III Secretion System), NOD1 and NOD2 (sensing peptidoglycan), AIM2 (sensing DNA), and Caspase-11/NLRP3 (sensing LPS).In a recent study published in Nature, Xu et al.⁴ describe the sensing of another pathogen-associated dynamic signal: the inactivation of Rho GTPases, which are molecular switches that regulate actin dynamics, by bacterial toxins. The authors identify Pyrin as the receptor responsible for detecting Rho GTPase inactivation. Mutations in the Pyrin-encoding gene MEFV have long been known to be the cause of the autoinflammatory disease familial Mediterranean Fever; the ligand for MEFV, however, had remain elusive until now. Using an array of Rho GTPase-targeting toxins from multiple bacteria, including the Clostridium difficile TcdB toxin and the C. botulinum C3 toxin, the authors show that Pyrin senses the inactivation of RHOA/B/C, but not other Ras GTPases. Since a variety of toxin-mediated Rho GTPase modifications, including glucosylation, adenylylation, ADP-ribosylation and deamidation, could trigger Pyrin activation, and no direct binding of the modified GTPases to Pyrin was observed, the authors conclude that Pyrin likely senses the effects of GTPase inactivation on the actin cytoskeleton rather than a specific modification itself.Because Rho GTPases control multiple fundamental cellular processes including cell motility, cytoskeletal morphology, and cell growth, they are highly attractive targets for manipulation by pathogenic bacteria^5,6. Both inactivation and activation of Rho GTPases by bacterial toxins or effector proteins have been well described; the former often results in inhibition of (immune) cell migration, disruption of the actin cytoskeleton and eventually cell death, while the latter often is utilized by bacteria to gain access into the cytosol. With the discovery of Pyrin-mediated detection of GTPase inactivation, and the recently described NOD1-mediated sensing of GTPase activation³, both types of “unnatural” actin dynamics are now known to be patrolled by the innate immune system. While Pyrin displays strict specificity for RHOA/B/C, NOD1 was shown to be more promiscuous and could detect activated RHOA, Rac1 and Cdc42.The findings by Xu et al. also raise several interesting questions. For instance, what exactly does Pyrin sense? As the receptor lacks a ligand-binding leucine-rich repeat domain, elucidating the function of the C-terminal B30.2 domain, which has been reported to bind actin and to facilitate co-localization with the cytoskeleton⁷, will likely shed light on Pyrin''s activation mechanism. Also, it will also be interesting to investigate whether certain pathogenic bacteria intentionally activate Pyrin-mediated inflammatory responses to provide a competitive advantage over resident commensals, as has been described for the inflammation induced during S. Typhimurium infection⁸. Finally, while a previous report has described that TcdB toxin from C. difficile activated an unknown inflammasome⁹, this study showed that the enzymatic activity of TcdB was dispensable. Further investigation will be required to clarify this discrepancy.“Effector-triggered immunity” has long been recognized as a central mechanism of immune-mediated detection of pathogens in plants¹⁰. The discovery that Pyrin can mediate a version of “effector-triggered immunity” in mice highlights the emerging similarities between pathogen detection by inflammasomes in mammals and “effector-triggered immunity” in plants. Future studies may reveal novel “effector-triggered immunity”-related functions for the remaining orphan NLRs.     

Comparison of avalanche survival patterns in Canada and Switzerland

Background

Current recommendations for rescue and resuscitation of people buried in avalanches are based on Swiss avalanche survival data. We analyzed Canadian survival patterns and compared them with those from Switzerland.

Methods

We extracted relevant data for survivors and nonsurvivors of complete avalanche burials from Oct. 1, 1980, to Sept. 30, 2005, from Canadian and Swiss databases. We calculated survival curves for Canada with and without trauma-related deaths as well as for different outdoor activities and snow climates. We compared these curves with the Swiss survival curve.

Results

A total of 301 people in the Canadian database and 946 in the Swiss database met the inclusion criteria. The overall proportion of people who survived did not differ significantly between the two countries (46.2% [139/301] v. 46.9% [444/946]; p = 0.87). Significant differences were observed between the overall survival curves for the two countries (p = 0.001): compared with the Swiss curve, the Canadian curve showed a quicker drop at the early stages of burial and poorer survival associated with prolonged burial. The probability of survival fell quicker with trauma-related deaths and in denser snow climates. Poorer survival probabilities in the Canadian sample were offset by significantly quicker extrication (median duration of burial 18 minutes v. 35 minutes in the Swiss sample; p < 0.001).

Interpretation

Observed differences in avalanche survival curves between the Canadian and Swiss samples were associated with the prevalence of trauma and differences in snow climate. Although avoidance of avalanches remains paramount for survival, the earlier onset of asphyxia, especially in maritime snow climates, emphasizes the importance of prompt extrication, ideally within 10 minutes. Protective devices against trauma and better clinical skills in organized rescue may further improve survival.A valanches make winter outdoor travel in mountainous terrain a hazardous activity. A total of 881 people died from avalanches in open terrain in Europe and North America over the six winters from 2003/04 to 2008/09.¹ The survival pattern of complete avalanche burials (coverage of the person’s head and chest, impairing breathing) in open terrain in Europe has been depicted in the avalanche survival curve,^2,3 which displays probability of survival as a function of burial time. The curve exhibits a characteristic shape, with four distinct phases. The probability of survival remains above 91% during the first 18 minutes of burial (“survival phase”). This phase is followed by a precipitous drop to 34% between 19 and 35 minutes because of asphyxiation of most people (“asphyxia phase”). Between 35 and 90 minutes, the survival curve levels out (“latent phase”) because of the survival of people with patent airways.⁴ Thereafter, survival drops again as those buried eventually succumb to lethal hypothermia complicated by progressive hypoxia and hypercapnia.⁵This avalanche survival model forms the foundation for current international recommendations for rescue and resuscitation^3,4 as well as the rationale for safety and rescue devices.⁶ However, the existing survival curve was calculated solely from Swiss data. Therefore, the universal validity of the survival curve and recommendations derived from it remains unknown.We analyzed survival curves for Canada and compared them with the survival curve in Switzerland. A better understanding of the factors affecting survival during an avalanche burial will provide important background for improvements in rescue, resuscitation and avalanche safety measures in Canada and elsewhere.     

In-cell infection: bringing uninvited guests

Cell-in-cell structures resulting from live cell engulfment were identified more than 100 years ago, but their physiological significance has remained largely obscure. Now Ni et al. identify a new role for cell-in-cell structure formation, called “in-cell infection” that spreads Epstein-Barr virus from infected B cells to epithelial cells, an activity that may predispose to cancer.Epstein-Barr virus (EBV) is a common herpesvirus infecting up to 90% or more of the human population that causes mononucleosis, is associated with autoimmune conditions, and predisposes to cancer¹. EBV persists as a latent infection within B cells and predisposes to cancers of B cell origin, including Hodgkin''s and Burkett''s lymphoma, due to expression of latency genes, which leads to B cell transformation. Infected individuals are also predisposed to developing nasopharyngeal and gastric carcinoma, as epithelial cells also harbor latent EBV. However, while the mechanism of EBV entry into B cells is well characterized, how EBV infects epithelium has remained obscure. In a recent paper published in Cell Research, Ni et al.² identify a novel mechanism for EBV infection of epithelial cells, which they term “in-cell infection”, an insidious mode of viral entry that takes advantage of whole cell ingestion.Viral infection is generally mediated by viral envelope glycoproteins that bind to specific receptors on target cells, leading to membrane fusion and viral entry. To infect B cells, the EBV envelope protein gp350 binds to the complement receptor 2 (CR2) on target cells, followed by interaction of gp42 with MHC class II molecules, and virus-to-target cell fusion is mediated by gp42, gH and gL proteins³. Unlike B cells, epithelial cells do not normally express complement receptors or MHC class II molecules, and are generally not infected by purified EBV. Previously described alternative modes of EBV infection of epithelial cells include “cell-to-cell” and “transfer” infection, where B cells have been found to act as carriers to mediate infection through cell adhesion protein-dependent conjugation^3,4,5,6.Ni et al.² now describe a different mode of epithelial cell infection, called “in-cell infection”, that occurs by ingestion of whole EBV-infected B cells, leading to the formation of “cell-in-cell” structures. B cell ingestion in this context resembles “entosis”, a mechanism previously found to mediate cell-in-cell structure formation in epithelial cultures and human tumors⁷. Entosis also promotes the uptake of hematopoietic cells into epithelial cells or cancer cells of various types^8,9. Incredibly, the authors find that entosis-like internalization of latent EBV-infected B cells (Akata) into cultured nasopharyngeal carcinoma cells (CNE-2) leads to the activation of EBV and the transfer of virus to host (CNE-2) cells. Cells infected in this manner express viral gene products, and produce virions upon stimulation that can infect naïve cells of either B cell or epithelial cell origin, indicating potent infection ability and altered tropism of EBV produced by this mechanism.Frequent cell-in-cell structure formation involving EBV-infected B cells is shown by the authors to occur in clinical nasopharyngeal carcinoma samples, suggesting that the in-cell infection mechanism is a likely contributor to viral spread in vivo, and may be linked to carcinoma development. Intriguingly, entosis itself may participate in tumorigenesis by promoting aneuploidy¹⁰, and by supplying cancer cells with nutrients¹¹. As the authors found that EBV infection promoted entosis-like cell uptake, this mode of viral spread could affect tumorigenesis by multiple mechanisms. For in-cell infection, it seems that the nutrients taken in upon the death of internalized cells come mixed with virus that is insidiously transferred to hosts, in a manner perhaps like a Trojan horse enterring with a hidden viral payload (Figure 1).Open in a separate windowFigure 1In-cell infection delivers virus to insusceptible host cells. The B cell infected by EBV resembles a Trojan horse that delivers a hidden viral payload to host epithelial cells.The identification of in-cell infection by Ni et al.² makes a significant contribution to cell-in-cell research by identifying a new pathophysiological role for an entosis-like process. Cell-in-cell structures were first reported over 100 years ago, but the mechanisms that control the formation of such structures and their significance are only now starting to emerge¹². As is often the case with groundbreaking research, the discovery of in-cell infection² raises many new interesting questions. What is the mechanism of B cell internalization into epithelial cells? Entosis is previously described to involve cell adhesion receptors, such as E-cadherin, and Rho-kinase that promotes the actomyosin contraction that drives cell uptake⁷. The molecular mechanism controlling the entry of EBV-infected B cells into epithelial cells will be important to uncover, as other mechanisms in addition to entosis can also mediate the uptake of live cells¹². How is EBV activated by the formation of cell-in-cell structures? How is EBV transferred from internalized cells to hosts? And importantly, can other viruses, such as HIV, spread by in-cell infection? The answers to these questions await further research.     

Defining and Mapping Mammalian Coat Pattern Genes: Multiple Genomic Regions Implicated in Domestic Cat Stripes and Spots

Mammalian coat patterns (e.g., spots, stripes) are hypothesized to play important roles in camouflage and other relevant processes, yet the genetic and developmental bases for these phenotypes are completely unknown. The domestic cat, with its diversity of coat patterns, is an excellent model organism to investigate these phenomena. We have established three independent pedigrees to map the four recognized pattern variants classically considered to be specified by a single locus, Tabby; in order of dominance, these are the unpatterned agouti form called “Abyssinian” or “ticked” (T^a), followed by Spotted (T^s), Mackerel (T^M), and Blotched (t^b). We demonstrate that at least three different loci control the coat markings of the domestic cat. One locus, responsible for the Abyssinian form (herein termed the Ticked locus), maps to an ∼3.8-Mb region on cat chromosome B1. A second locus controls the Tabby alleles T^M and t^b, and maps to an ∼5-Mb genomic region on cat chromosome A1. One or more additional loci act as modifiers and create a spotted coat by altering mackerel stripes. On the basis of our results and associated observations, we hypothesize that mammalian patterned coats are formed by two distinct processes: a spatially oriented developmental mechanism that lays down a species-specific pattern of skin cell differentiation and a pigmentation-oriented mechanism that uses information from the preestablished pattern to regulate the synthesis of melanin profiles.PATTERNED coats are typical of many mammalian groups, whose spots, stripes, and other markings have been hypothesized to play important adaptive roles in camouflage, predator evasion, and social communication (Cott 1940; Searle 1968; Ortolani and Caro 1996). Many mammals bear striped or spotted coats, and these phenotypes have historically drawn attention from many fields of human science and culture (e.g., the leopard''s spots, or the stripes seen in tigers and zebras). Although several theoretical studies have proposed mathematical models that could underlie the developmental dynamics of coat pattern formation in mammals (Murray and Oster 1984; Oyehaug et al. 2002), no direct investigation of the genetic basis of these phenotypes has yet been performed, so that their mechanistic causes remain a mystery. Recent advances in genomics, molecular biology, and evolutionary developmental biology (Evo-Devo) have revealed genes and pathways involved in skin pattern formation in Drosophila (Schug et al. 1998; Gompel et al. 2005; Prud''homme et al. 2006; Parchem et al. 2007), butterflies (Joron et al. 2006a,b), and zebrafish (Iwashita et al. 2006; Watanabe et al. 2006; Svetic et al. 2007). In contrast, despite the relevance of characterizing equivalent processes in mammals, little progress toward this goal has been accomplished, perhaps due to the lack of adequate mammalian models exhibiting variation in skin pattern and for which genetic and genomic tools were available.The domestic cat is a very promising model in this regard, as it presents several coat pattern variants and a growing body of genetic and genomic tools suitable for gene identification (Menotti-Raymond et al. 2003; Murphy et al. 2007; Pontius et al. 2007; Pontius and O''Brien 2007; Davis et al. 2009). Classic work on domestic cat coat color (Robinson 1958; Lomax and Robinson 1988) has suggested that there is a monogenic allelic series of coat patterns in the domestic cat, controlled by the Tabby (T) locus: in order of dominance, the four recognized alleles would be Abyssinian or “ticked” (T^a), Spotted (T^s), Mackerel (T^M), and Blotched (t^b) (Figure 1). Although there has been little doubt among breeders that the “mackerel” and “blotched” forms segregate as a single autosomal locus, this may not be the case for the other two phenotypes (T^a and T^s), which so far have not been tested thoroughly for allelism relative to the more common Tabby variants T^M and t^b. Some breeding data have suggested that these variants may not be allelic with the main Tabby locus (Lorimer 1995), but further scrutiny is required to test this hypothesis. A recent genetic study (Lyons et al. 2006) considered the Abyssinian variant as an allele of Tabby, reflecting the prevalent perception that they are coded by the same locus. Testing this hypothesis, and identifying the implicated genomic region (or regions), is a first step in the process of dissecting the molecular and developmental basis for these pattern-formation phenotypes.Open in a separate windowFigure 1.—Major coat pattern phenotypes of the domestic cat (Felis silvestris catus). A “hierarchy” of pelage patterns is observed in this species, with the absence of markings seen in Abyssinian cats (A) dominating over a spotted coat (B), which dominates over a “mackerel” (striped) coat (C), itself dominant over the blotched pattern (D). The classical, single-locus model for this phenotypic variation proposed the allelic series T^a > T^s > T^M > t^b for these respective variants.Aiming to investigate the genetic basis of pattern formation on the domestic cat pelage by genomic, positional methods, we established three separate pedigrees segregating for different combinations of coat pattern phenotypes. Our results demonstrate that at least three different loci underlie the striping and spotting patterns observed in domestic cats and identify the genomic location of two of them.     

Rainy weather and medical school admission interviews

Mood can influence behaviour and consumer choice in diverse settings. We found that such cognitive influences extend to candidate admission interviews at a Canadian medical school. We suggest that an awareness of this fallibility might lead to more reasonable medical school admission practices.Admission offers to medical school are competitive and sometimes based on an interview. Psychology research suggests, however, that interviews are prone to subconscious biases from extraneous factors unrelated to the candidate.¹ One of the most fundamental observations is that people interviewed on rainy days tend to receive lower ratings than people interviewed on sunny days.² We studied whether this bias also extends to admission interviews at a large Canadian medical school.We analyzed the results of consecutive medical school interviews at the University of Toronto between 2004 and 2009. We included all data available with no exclusions. Almost all interviews occurred in the early spring. Scores for each interview were obtained from the admissions office as recorded from 0 to 20.³ This Likert scale was anchored with integer values where 10 denoted “unsuitable,” 12 denoted “marginal,” 14 denoted “fair,” 16 denoted “good,” 18 denoted “excellent” and 20 denoted “outstanding.”We obtained weather data from the official government archive and defined a priori the day as “rainy” if precipitation (including freezing rain, snow and hail) occurred in the morning or afternoon.⁴ Otherwise, we defined the day as “sunny.” We did not examine more complex combinations with time lags, such as when a sunny day followed multiple rainy days.A total of 2926 candidates were interviewed over the 6-year period. As expected, their demographic characteristics were unrelated to the weather (Appendix 1, available online at www.cmaj.ca/cgi/content/full/cmaj.091546/DC1). Overall, those interviewed on rainy days received about a 1% lower score than those interviewed on sunny days (average score 16.31 v. 16.49, p = 0.042). This pattern was consistent for both senior interviewers (16.39 v. 16.55, p = 0.08) and junior interviewers (16.23 v. 16.42, p = 0.041). We next used logistic regression to analyze subsequent admission decisions. The difference in scores was equivalent to about a 10% lower total mark on the Medical College Admission Test.Open in a separate windowWe suggest that cognitive patterns evident in controlled psychology laboratories can also occur in regular medical settings. The magnitude of the specific influence may be modest, but such small differences can be important in some cases because each year there are about 100 candidates who receive a score within 1% of the admission threshold.⁵ In this study, we examined only one extraneous influence on mood. Many additional factors may also affect mood (e.g., ambiance, deportment, humour and scent).² Calling attention to these issues may diminish their impact on judgment.¹